*3.1.1 Riboflavin*

Riboflavin, also known as vitamin B2, is a dimethylated isoalloxazine linked to ribitol. In fermented beverages like wine, this heterocyclic ring is mainly found as free riboflavin although few amounts of the mononucleotide and dinucleotide forms can be also found. This is why some analytical methods propose to convert these forms to free form prior the analysis and to quantitate the total riboflavin content [26]. Traditionally, this compound has been determined by microbiological or fluorimetric methods [60]. However, when dealing with complex samples such as wine, the high performance liquid chromatography with reversed-phase column (RP-HPLC) is the most suitable technique as it separates the riboflavin from interferences. Among the different possible detectors including UV/vis, mass spectrometry and fluorescence, the latest is the most used because riboflavin naturally fluoresce. This property allows the injection of the sample directly into the HPLC although a sample filtration is recommended to avoid light scattering effect [26, 61]. Finally, it should be noted that some alternatives to these expensive and time-consuming HPLC methods have been developed. One of them is based on the fluorescence quenching effect produced by the riboflavin-binding protein what is measured by using a single diode fluorimeter [62]. According to the authors, the results obtained are comparable to those obtained by HPLC methods. The other alternative involves the use of UPLC which has become the modern HPLC showing higher sensitivity and chromatographic efficiency with a consequent run-time decrease [63].
